Molecular Diagnostics in Thyroid Tumors

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1 USCAP 2011 Endocrine Pathology Society Companion Meeting Molecular Diagnostics in Thyroid Tumors Yuri E. Nikiforov, M.D., Ph.D. Department of Pathology University of Pittsburgh Medical Center

2 Outline Overview of genetic alterations in thyroid cancer Diagnostic mutational markers in thyroid nodules Prognostic mutational markers for thyroid cancer

3 TSHR RTK Well Differentiated Cancer Poorly Differentiated/AC β α γ RET/PTC Adenylate cyclase RAS PI3K camp PTEN PKA MAPK pathway BRAF MEK AKT mtor PI3K/AKT pathway ERK PAX8/PPARγ TP53

4 Somatic Mutations in Thyroid Cancer NRAS NTRK1 TP53 PIK3CA PPARγ AKT1 Intrachromosomal rearrangenement Interchromosomal rearrangenement KRAS Point mutation HRAS PTEN RET BRAF BRAF

5 Frequency of Mutations in Thyroid Cancer Papillary Carcinoma Follicular Carcinoma None RAS 15% 45% 15% RET/PTC BRAF None BRAF RAS RET/PTC None 40% 30% PAX8/PPARγ RAS RAS 75% of tumors with known mutations 70% of tumors with known mutations

6 BRAF Mutations 464 Gly 466 Gly Activation P loop loop RBD C Kinase domain Val Glu Gly Leu Phe Gly Leu Ala The Val Lys Ser K601E VK600-1E V599Ins V600D- FGLAT ins T599I-VKSR600-3del AKAP9/BRAF P T1799A V600E T1799A V600E P inv(7)(q21-22q34) 98-99% 1-2% Prevalence: 40-45% in PTC and 20-30% in PDC/AC

7 RET/PTC Rearrangement RET EC TK H4 RIα NCOA4 Chromosome 10 RET receptor Frequency of RET/PTC types TK RET RET/PTC1 RET/PTC2 RET/PTC3 and novel types 70% <3% 30%

8 Prevalence and Specificity of RET/PTC Rearrangement Clonal RET/PTC: present in >1% of tumor cells 20* 21* * 47* 48 76* 77* 6.3kb EcoRI Standard Sensitivity RT- PCR FISH with appropriate cut-off levels Southern Blot Non-clonal RET/PTC: present in <1% of tumor cells 77 (60,000 cells) 24 (13 cells) 25 (68 cells) 28 (1.7cells) 32 (1.8 cells) 47 (5,000 cells) 46 (2,000 cells) 43 (2 cells) PC NC RET/PTC1 High Sensitivity RT-PCR 76 (60,000) 62 (23) 64 (3.8) 66 (4.3) 68 (14) PC NC RET/PTC3 Zhu et al. JCEM 2006,91:

9 Prevalence and Specificity of RET/PTC Detection Prevalence of clonal RET/PTC: Adults 10-20% ( reported range 0-80%) Children, sporadic 40-50% Children, h/o radiation 50-80% Specificity: Clonal rearrangement specific for papillary CA Non-clonal rearrangement found in other lesions

10 RAS Mutations Point mutations Most common hotspots in thyroid: NRAS codon 61 HRAS codon 61 KRAS codons 12/13 Goiter Follicular Follicular Papillary PDCA adenoma Carcinoma Carcinoma and AC Range 0-20% 20-45% 30-50% 0-20%* 20-90% Average 5% 30% 40% 15% 30% *Almost all are follicular variants of PTC Meta-analysis of literature

11 PAX8/PPARγ Rearrangement t(2;3)(q13;p25) Fusion involves PAX8 and PPARγ genes Goiter Follicular Follicular Papillary PDCA adenoma Carcinoma Carcinoma and AC Range % 20-70% 0-25% 0 Average 0 7% 30% 5% 0 * A single case of CREB3L2-PPARγ fusion in follicular carcinoma reported (Lui WO et al., Cancer Res 2008) Meta-analysis of literature

12 Diagnostic Molecular Markers I. Single mutation: BRAF RET/PTC RAS II. Panel of mutations: BRAF, RET/PTC, RAS, PAX8/PPARg

13 Management of Patients with Thyroid Nodules Amenable to FNA? TSH Suppressed TSH Unsuppressed Cold Nodule Present? FNA cytology Ultrasound Guided FNA

14 Thyroid Nodules and FNA Cytology Bethesda Classification Diagnostic Category Risk of malignancy Benign <3% Malignant >98% Indeterminate cytology Follicular lesion of undetermined significance (FLUS) 5-10% Follicular neoplasm/lesion 20-30% Suspicious for malignancy 50-75% Nondiagnostic Baloch ZW et al. Diagn Cytopathol 2008;36:425-37

15 BRAF V600E is Highly Specific for Malignancy Reported Studies Specificity of BRAF in Thyroid FNAs Samples (n) BRAF-positive (n) Final Surgical Pathology Diagnosis 9 FNA Prospective PTC: 159 (100%) 7 FNA retrospective PTC: 291 (100%) 2 Research FNAs PTC: 130 (99.2%) HN: 1 (0.8%) Total: PTC: 580 (99.8%) HN: 1 (0.2%) Nikiforova MN & Nikiforov YE Thyroid (2009)

16 Testing for a Panel of Mutations in Thyroid FNA Samples BRAF V600E K601E RAS NRAS codon 61 HRAS codon 61 RET/PTC RET/PTC1 RET/PTC3 PAX8/PPARγ KRAS codons 12/13 Controls: Quality and quantity of DNA and RNA Quantity of epithelial cells

17 Performance of Mutational Panel in Thyroid FNA Samples Prospective FNA Study Nikiforov et al, JCEM 2009 Cantara et al, JCEM 2010 Nikiforov et al, (submitted) Location Cincinnati, Denver USA Siena Italy Pittsburgh USA Number of FNA Samples/ Sample Type Number of Mutations Identified 470 FNAs consecutive, all 32 (7%) types 285 FNAs from patients with surgery 1149 FNAs consecutive, indeterminate and positive cytology 67 (29%) 126 (11%)

18 Performance of Mutational Panel in Thyroid FNA Samples Prospective FNA Studies Nikiforov et al, JCEM 2009 Cantara et al, JCEM 2010 Nikiforov et al, ((submitted) Probability of Cancer in Nodules Positive for Specific Mutations BRAF (n=123) RAS (n=79) RET/PTC (n=20) PAX8/PPARγ (n=5) 100% 87% 100% 100% 100% 74% 100% - 100% 84% 100% 100% TOTAL 100% 83% 100% 100%

19 Molecular RAS Mutations Cytology Histology RAS+ (71) Surgery (67) Indeterminate (64) FLUS (19) FN/FL (25) Suspicious (10) PTC (52) FTC (4) FA (11) 84% 16% Negative (3) PTC (1) FTC (1) FA (1)

20 RAS Mutations in Follicular Variant PTC MUT WT NRAS 61 MUT WT NRAS 61 MUT WT NRAS 61

21 Molecular Testing in FLUS Cytology 513 samples Molecular Histology 124 (24%) FLUS 117 (94%) acceptable for molecular analysis Positive 12 (10%) RAS - 8 BRAF - 3 Negative 105 (90%) PAX8/PPARg /12 (100%) 8/105 (7.6%) Ohori et al. Cancer Cytopathology (2010)

22 Revised Management Guidelines for Patients with Thyroid Nodules and Differentiated Thyroid Cancer American Thyroid Association, 2009

23 Prognostic Molecular Markers

24 BRAF V600E Is Associated with Tumor Recurrence and Mortality All Variants of PTC included Conventional PTC Only All Variants of PTC included N=219 N=203 N=102 Xing M et al. JCEM (2005) Kim TY et al. Clin Endocrinol Elisei R et al. JCEM (2008) (2006) BRAF + (n=107) BRAF (n=112) Extrathyroidal Extension 41% 16% <0.001 LN Metastases 54% 21% <0.001 Tumor Stage III or IV 30% 14% p

25 BRAF V600E Predisposes to Tumor Dedifferentiation WD PTC PDC DNA DNA BRAF V600E BRAF V600E Nikiforova M et al. JCEM (2003)

26 BRAF V600E Is Associated with 131 I-Refractory Disease Mutational frequency of BRAF, RET/PTC, NRAS, HRAS, KRAS, AKT1, and PIK3CA in (A) 18 primary ATC, (B) 34 primary PDTC, and (C) 23 RAIR, FDG-PET-positive PDTC. Ricarte-Filho JC et al. Cancer Res 2009;69:

27 Should BRAF-positive PTCs be Treated Differently? Retrospective review 106 consecutive patients with BRAF+ PTC compared to a cohort of 100 BRAF- patients 75 BRAF+ patients had unknown BRAF status preoperatively 12 received lobectomy then required completion 6 required re-operation for persistent disease within 2 years Yip L et al. Surgery 2009;146:1215

28 Should BRAF-positive PTCs be Treated Differently? 18 of 75 patients (24%) would have received different treatment had BRAF status been know pre-operatively BRAF-positivity more than triples the need for re- operation within the first 2 years of follow up Yip L et al. Surgery 2009;146:1215

29 Potential BRAF Impact on Patient Management Initial surgical management of PTC, including microcarcinoma - Total thyroidectomy, possible central compartment l/n dissection Initial radioiodine treatment - Higher dose of iodine Follow-up of patients - TSH level in low subnormal range Xing M. Mol Cell Endocrinol (2005)

30 Papillary Thyroid Microcarcinoma (PTMC) WHO Definition: Papillary carcinoma 1 cm or less in size Incidental finding Incidence in autopsy series: % in North America 19-28% in Japan 36% in Finland

31 Papillary Microcarcinoma and Patient Outcome Some PTMCs are Aggressive Publication Patient No. (Follow-up) Lymph Node Metastases Distant Metastases Tumor Recurrence Cancer- Related Deaths Hay et al. (1992) Baudin et al. (1999) Sugitani et al. (1999) Ito et al. (2003) Roti et al. (2006) 535 (16 y) 281 (7.3 y) 178 (10.7 y) 732 (1.6-4 y) 243 (5.1 y) 32% 0.4% 5% 0.4% 43% 2.8% 3.9% 0 ND 2.2% ND 2.2% 48% 0 2.6% 0 13% 1.6% 1.6% 0 Summary of 11 studies % (12-70%) 0.7% (0-2.8%) 5% (1.6-26%) 0.3% (0-2.2%) Mazzaferri EL. Endocr Pract (2007)

32 Molecular and Histopathological Features of Aggressive and Non-Aggressive PTMCs AGGRESSIVE PTMCs N= 29 NON-AGGRESSIVE PTMCs N= 30 P VALUE BRAF 20/26 (77%) 8/25 (32%) ANATOMIC LOCATION - LEFT LOBE/ RIGHT LOBE 15 (52%) / 14 (48%) 14 (47%) / 14 (47%) SUPERFICIAL TUMOR LOCATION 27 (93%) 9(30%) < POSITIVE MARGIN 2 (7%) 1 (3%) COMPLETE CAPSULE 2 (7%) 8 (27%) INFILTRATIVE BORDER 29 (100%) 24 (80%) MICROSCOPIC VARIANT - CLASSIC PAPILLARY 13 (45%) 11 (37%) FOLLICULAR VARIANT 8 (28.0%) 18 (60%) TALL CELL FEAT/VAR 8 (28%) 1 (3%) INTRATHYROIDAL SPREAD/MF 26 (90%) 13 (43%) < TUMOR FIBROSIS - NO 2 (7%) 9 (30%) YES, (83%) 12 (40%) EXTRATHYROIDAL EXTENSION 19 (66%) 4 (13%) < Niemeyer LA et al. (submitted)

33 Statistical Analysis: Multivariate Regression Model Estimate Std. Error Z value P value BRAF mutation * Superficial tumor location Intraglandular spread/multifocality * * Fibrosis (2+) MP Score = 3x BRAF + 4x Superf. location + 3x IGS/MF + 2x Fibrosis ( 2+) Niemeyer LA et al. (submitted)

34 Histopathologic Features of Aggressive Papillary Thyroid Microcarcinomas Superficial Location Fibrosis 2+ Niemeyer LA et al. (submitted)

35 Histopathologic Features of Aggressive Papillary Thyroid Microcarcinomas Intraglandular spread/multifocality Isolated psammoma body Tumor foci in normal thyroid tissue adjacent to Lymphatic invasion main tumor nodule Niemeyer LA et al. (submitted)

36 Molecular-Pathological Score To Define TPMC Aggressiveness MP Score BRAF Mutation + Superficial (subcapsular) location Intrathyoidal spread/mf Tumor fibrosis, 2+ x3 x4 x3 x2 Validation in independent set of 40 TPMCs 70% 25% 5% 30 Aggressive Control group Not Aggressive aggressive TPMCs Low risk (0%) MP Score Moderate risk (27%) High risk (67%) Niemeyer LA et al. (submitted)

37 Conclusions And Future Directions Diagnostic use of mutational markers: High PPV for cancer (close to 100%, except RAS ~85%) Need to define distinct algorithms of patient management based on cytology + molecular analysis Prognostic mutational markers: BRAF V600E tumors - surgical and post-surgical management Additional markers of tumor aggressiveness Future directions: Discovery of new mutations in thyroid cancer to completely resolve uncertainty of indeterminate cytology